Endostatin Is Increased By Knockdown Of sVEGFR-1 In The Mouse Cornea

Abstract

Purpose: : The cornea is normally avascular to permit visual clarity. However, in disease, neovascularization (NV) can occur and compromise clarity and vision. Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis in many models, including the cornea. Soluble form of VEGF receptor-1 (sFlt-1) has been shown to be anti-angiogenic in cornea. There are other inhibitors of angiogenesis in the cornea, include endostatin, and pigment epithelium-derived factor (PEDF). Here, we investigate whether these anti-angiogenic factors are affected by sFlt-1 modulation in the cornea.

Methods: : sflt-1 was specifically down-regulated by RNA interference (RNAi). We injected into the BALB/C mice’s corneas a plasmid expressing a short hairpin RNA (shRNA) targeted against a sequence in the unique carboxyl-terminus region of sflt-1 (pshRNA-sflt-1). The controls were PBS and a plasmid which expressed a nonspecific shRNA (pSEC-negative). RT-PCR, quantitative RT-PCR were performed in corneas from PBS, pSEC-negative and pshRNA.sflt-1 injection groups (n=3) at Day 2, Day 4, and Day 7 three time points.

Results: : Corneal neovascularization was induced by pshRNA-sflt within 7 days of injection (n=7), but neither by pSEC-negative nor PBS. Quantitative RT-PCR data showed that endostatin was upregulated, relative to control (nonspecific plasmid-mediated shRNA) by 250% at Day 2, 400% at Day 4, and 270% at Day 7 (all p’s <0.05). However, PEDF was decreased by 51% (P< 0.01) at Day 2 compared to control.

Conclusions: : Suppression of sflt-1 is accompanied by increase of mRNA of endostatin after pshRNA-sflt-1 induced corneal neovascularization. The mechanism in which these factors work merits further investigation.

Keywords: neovascularization • vascular endothelial growth factor • cornea: basic science